The combination of genetically engineered bacterial cells and recombinant plasmids are the foundation of industrial biotechnology. For example, they are used for production of industrially and medically important proteins such as enzymes, cytokines, growth hormones, and antigens or as live bacterial vaccines. The advent of DNA immunization and gene therapy technologies has added another dimension to the use of genetically engineered bacterial cells and their companion recombinant plasmids. For the purpose of these technologies, expression of proteins in genetically engineered bacterial cells is no longer the principal objective, which is, instead, the replication and high-level production of structurally and genetically stable recombinant plasmids carrying foreign DNA. This is because the DNA, rather than a protein encoded therein, is the desired product for use in DNA immunization or gene therapy.
Consequently, the present invention relates to the use of genetically-engineered bacterial cells and their companion recombinant plasmids for cloning of foreign DNA. More particularly, the invention provides a method whereby foreign DNA suitable for use in DNA immunization and gene therapy can be replicated and produced in large quantities in these companion recombinant plasmids.
Early on during the development of recombinant DNA technology, it was realized that a major challenge to that emerging technology was the stable maintenance of recombinant plasmids in bacterial cells. It was also realized that this problem stems largely from the heavy metabolic burden imposed on genetically-engineered bacterial cells as a result of high-level expression of proteins which are of no value to them.
Consequently, when propagating genetically-engineered bacterial cells that have no incentive to maintain the recombinant plasmid, over time, plasmid-free bacterial cells appear at increasing frequency. Because of the heavier metabolic burden on plasmid-harbouring bacterial cells, plasmid-free bacterial cells have higher growth rates. Accordingly, within a relatively short period of time, the bacterial culture can become dominated by plasmid-free bacterial cells, thus leading to a decreasing plasmid yield.
Since plasmid-harbouring bacterial cells are almost always at a growth disadvantage as compared to plasmid-free bacterial cells, any plasmid-free bacterial cells which arise during extended culture periods will eventually take over the fermentation bioreactor. In this regard, it is estimated that a 10% growth advantage for plasmid-free bacterial cells will result in bioreactor take-over within 150 hours of culture at a dilution rate of 1 hr/L even if the plasmid loss frequency is only 1.times.10.sup.-7. These calculations underscore the need for a plasmid stabilization system that is 100% effective in preventing plasmid loss since it is not uncommon that bacterial cells are grown for 300 hours of continuous culture within industrial bioreactors.
These observations indicate that absence of selective pressure for maintaining recombinant plasmids in propagated genetically engineered bacterial cells results in a decreasing frequency of plasmid-harbouring bacterial cells, and that plasmid loss is further accentuated by the slower growth rate of plasmid-harbouring bacterial cells.
Several methods have been devised to enhance recombinant plasmid stability in bacterial cell populations. All these methods have a common underlying principle: the application of selective pressure to ensure the growth and multiplication of only those bacterial cells that are harbouring the recombinant plasmid.
In one of these methods, selective pressure is applied to bacterial cells by cloning the desired gene on a recombinant plasmid that also carries one or more genes specifying resistance to specific antibiotics. Thus, addition of the specific antibiotic(s) to a culture of growing bacterial cells ensures that only the ones harbouring the recombinant plasmid survive.
Although antibiotic resistance genes have been very useful and effective in providing a means of recombinant plasmid stability, their use has serious drawbacks. Firstly, addition of antibiotics in the culture medium during fermentation on an industrial scale is expensive. Secondly, in those cases where the mechanism of antibiotic resistance is based on secretion of an antibiotic-inactivating compound, some plasmid-free bacterial cells may survive because surrounding plasmid-harbouring bacterial cells secrete enough of the antibiotic-inactivating compound into the culture medium so as to permit survival of both plasmid-harbouring and plasmid-free bacterial cells. Thirdly, the use for DNA immunization and gene therapy of recombinant plasmid DNA containing antibiotic resistance genes is viewed unfavourably because such genes might be incorporated into the animal genome, or into the genome of endogenous microflora. Fourthly, residual antibiotics that contaminate plasmid DNA (as a result of their addition to the culture medium) could provoke sensitivity and/or systemic allergic reactions in certain animals treated with such plasmid DNA.
As an alternative to the use of antibiotic resistance genes, several methods have been devised to enhance recombinant plasmid stability and prevent accumulation of plasmid-free bacterial cells. The common thread to these methods is to make the survival of genetically-engineered bacterial cells dependent on a functional complementation system using a plasmid-borne gene.
These known methods can be divided into three groups depending on the type of polypeptide encoded by the gene used. However, each of the known methods is either impractical in reducing the rate at which plasmid-free bacterial cells arise under industrial conditions and/or unsuitable for production of recombinant plasmids for use in eukaryotes, such as for DNA immunization and gene therapy. Each group of methods will be described in turn.
The first group comprises methods in which a chromosomal defect results in the failure to produce an essential nutrient and the plasmid-borne gene encodes an enzyme essential for the biosynthesis of this nutrient (e.g. an amino acid) that normally exists in commonly used bacterial growth medium (Dwivedi, C. P. et al. Biotechnology and Bioengineering (1982) 24: 1465-1668; Imanaka, T. et al. J Gen Microbiol (1980) 118: 253-261). These methods as set forth in the prior art require that the plasmid-harbouring bacterial cells be grown on special and expensive synthetic medium lacking the amino acid in question. This is impractical in industrial bioreactor conditions.
The second group comprises methods wherein a chromosomal defect resides in defective production of an end-product that is required, and the plasmid-borne gene encodes an enzyme that synthesizes this end-product, but where the end-product does not exist in commonly used bacterial growth medium (Diderichsen, B. Bacillus Molecular Genetics and Biotechnology Applications (1986) pp. 35-46; Ferrari, E. et al. BioTechnology (1985) 3: 1003-1007; Galan, J. E. et al. Gene (1990) 94: 29; Nakayama, K. et al. BioTechnology (1988) 6: 696; Curtiss, R. et al. Res Microbiol (1990) 141: 797).
To date, this method has focused on synthesis of amino acids that are incorporated into the bacterial cell wall. The utility of this approach in DNA immunization and gene therapy is hampered for the following reasons.
The enzymes that have been used in these situations to date (e.g., aspartate semialdehyde dehydrogenase (asd) or alanine racemase (alr)) catalyse the formation of a small diffusible growth factor (aspartate semialdehyde and D-alanine, respectively), and the factor may accumulate in the culture medium under industrial bioreactor conditions. Such accumulation contributes to plasmid loss because of a cross-feeding effect in which plasmid-harbouring bacterial cells (producing the small diffusible growth factor) support the growth of plasmid-free bacterial cells. Therefore, to avoid such cross-feeding effects, this type of gene has been used with low-copy-number plasmids, i.e., of a type which occur as only 1 or 2 copies per bacterial cell. Clearly using low-copy-number recombinant plasmids is impractical for production of industrial quantities of plasmid DNA, where plasmids of a type resulting in high copy numbers are desired. High-copy-number recombinant plasmids are those of a type which occur somewhere on the order of about 50 to several hundreds of plasmid copies per bacterial cell.
The asd gene as the plasmid-borne gene has another drawback. This drawback is related to the recent findings (Park, J. T. Molecular Microbiology (1995) 17: 421-426) that bacterial cells (e.g. E. coli) actually degrade approximately 50% of their peptidoglycan layer. The degradation product is a tripeptide consisting of L-alanine/D-glutamate/mesodiaminopimelic acid, which is reused by the bacterial cells to form peptidoglycan thus conserving energy that the bacterial cell would have expended synthesizing new tripeptide components of peptidoglycan. A high proportion of this tripeptide is released into the culture medium and is available to be taken up by neighbouring bacterial cells for incorporation into their own peptidoglycan layer. The asd gene encodes the first enzyme in the biosynthesis of the amino acid mesodiaminopimelic acid (dap) which is already included in this tripeptide. However, since bacterial cells can recycle their own peptidoglycan, and can secrete the tripeptide containing the replaced amino acid dap, there is no selective pressure on the plasmid-harboring bacterial cells to maintain their plasmids.
The third group comprises methods wherein the plasmid-borne gene encodes a protein that has functional and structural counterparts in eukaryotic cells and/or is capable of acting upon a eukaryotic cell component. The use of such genes represents a major safety concern because of the potential for the proteins encoded by these genes to function in eukaryotic cells and because of the potential for integration of the gene itself into the eukaryotic genome by homologous recombination. Examples include genes encoding proteins involved in the vital function of DNA replication (single strand DNA binding protein; Porter, R. D. et al. BioTechnology (1990) 8: 47) or a tRNA-related function (valine tRNA synthetase; Nilsson, J. and Skogman, G. BioTechnology (1986) 4: 901-903).
The use as a marker of the gene encoding alanine racemase (alr) is also impractical for producing recombinant plasmid DNA for DNA immunization and gene therapy because this enzyme can function in eukaryotic cells. Alanine racemase catalyses the conversion of L-alanine into D-alanine. Since eukaryotic cells contain L-alanine as a natural component of their biochemical make-up, the use of alanine racemase can interfere with the biochemical reactions involved in L-alanine biosynthesis in eukaryotic cells and could lead to the formation of an amino acid (D-alanine) that does not naturally exist in eukaryotic cells.
Recently, a meeting of the World Health Organization (WHO) devoted to issues of DNA immunization and gene therapy was convened (Nucleic Acid Vaccines, WHO, Geneva, as reported in Cichutek, K. Vaccine (1994) 12: 1520; Robertson, J. S. Vaccine (1994) 12: 1526; Smith, H. Vaccine (1994) 12: 1515). At this meeting of experts in the field of DNA immunization and gene therapy, as well as experts from regulatory authorities, a number of matters were declared crucial issues that have to be addressed in order to pave the way for these technologies to produce clinically useful products. These included structural and genetic stability of recombinant plasmids, the potential integration of recombinant plasmid DNA within host chromosomes, as well as the use of marker genes (e.g. antibiotic resistance genes) for selection and propagation of the desired plasmid-harbouring bacterial cells.
Thus, for the purpose of introducing foreign DNA into eukaryotes, such as for DNA immunization and gene therapy, there is a need for a system whereby genetically-engineered bacterial cells can be used for production of plasmid-borne foreign genes without the use of genetic material which itself can integrate within the eukaryotic genome or by virtue of its encoded product can function within eukaryotic cells or act upon any eukaryotic cell component.
In addition, improved methods for stable and high-productivity cloning in bacteria would be helpful simply for production of large quantities of desired DNA.